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Applied Biochemistry and Microbiology (v.47, #4)
Plant growth promoting rhizobacteria as alternative to chemical crop protectors from pathogens (review) by I. V. Maksimov; R. R. Abizgil’dina; L. I. Pusenkova (pp. 333-345).
The review analyses data on physiological and biochemical influence of rhizospheric and endophytic plant growth promoting rhizobacteria (PGPR) on induced the mechanisms of resistance of plants and the possibility of their using in agricultural for to protect crop from pathogens and phytophages. Resistance of plants promoted by PGPR due to their endosymbiotic interrelationships is directly achieved by producing peptide antibiotics and hydrolases of chitin and glucan and also because plants form their own system of induced resistance, accompanied by changes in the balance of defensive proteins, phytohormones, and pro-/antioxidant status.
Antioxidant activity of oxygen-containing aromatic compounds by M. V. Potapovich; V. P. Kurchenko; D. I. Metelitza; O. I. Shadyro (pp. 346-355).
Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H2O2-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K i, μM) or the inhibition degree (%). K i values varied in the range of 4 to 500 μM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds of guaiacyl and syringal series exhibited high antioxidant activity (K i in the range of 10–300 μM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycoumaric acid.
Identification of a new bioregulator acting in ultralow doses in bulb onion (Allium cepa L.) by O. G. Kulikova; V. P. Yamskova; A. P. Il’ina; D. V. Margasyuk; A. A. Molyavka; I. A. Yamskov (pp. 356-360).
A bioregulator that has physicochemical and biological properties similar to a group of bioregulators isolated from various animal tissues has been found in the bulb onion (Allium cepa L.). It was determined that the biological action of the plant bioregulator is determined by a peptide with molecular weight of 4036 ± 2 Da whose 18-C-terminal amino acid sequence consisted of 18 residues. On models of seed germination of some vegetable cultures, the ability of the bioregulator isolated from supernatant of onion extract in ultralow doses (10−13 mg of protein/ml) to inhibit growth and development was demonstrated.
Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers by I. A. Parfenov; T. A. Revina; P. P. Pashkovsky; N. L. Radyukina; T. A. Valueva (pp. 361-365).
Product of polymerase chain reaction designated as PKPIJ-B was isolated after amplification from genomic DNA of potato (Solarium tuberosum L., Zhukov Jubilee cultivar) using the designed primers. Nucleotide sequence of PKPIJ-B was determined and amino acid sequence of protein was restored. Analysis of this sequence has allowed us to suggest that isolated gene fragment encodes chymotrypsin and trypsin inhibitor protein (PKCI-23 potato Kunitz-type chymotrypsin inhibitor) of potato tubers.
Over-expression, purification and functional characterization of Celosia ClpS as a fused protein in Escherichia coli by A. Gholizadeh (pp. 366-372).
A ClpS homologue from Celosia cristata was expressed as maltose-binding fusion protein under the control of strong inducible tac promoter of pMALc2X vector in TB1 strain of Escherichia coli. SDS-PAGE analysis showed that fused ClpS is produced as about 63 kDa protein in recombinant bacteria. Expressed product was purified to homogeneity with a yield of about 31 mg/l of bacterial culture. The results indicated that heterologous expression of Celosia ClpS does not affect bacterial growth under different induced conditions. Total cellular antioxidant assessment results revealed that the induction of ClpS activates the bacterial antioxidative system. Since, the purified ClpS did not exhibit antioxidant activity in vitro, we speculated a functional corelation between bacterial proteolytic apparatus and its anti-oxidative system. This prediction may contribute to our better understanding of functional relationship between proteolytic and antioxidative systems in biological worlds in the future investigations.
Anaerobic synthesis of succinic acid by recombinant Escherichia coli strains with activated NAD+-reducing pyruvate dehydrogenase complex by A. Yu. Skorokhodova; A. Yu. Gulevich; A. A. Morzhakova; R. S. Shakulov; V. G. Debabov (pp. 373-380).
Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+-reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acid from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids through deletion of the genes ackA, pta, poxB, and ldhA (SGM0.1) in E. coli MG1655 strain and by additional introduction of the Bacillus subtilis pyruvate carboxylase (SGM0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields of control strains by 2 and 33% in the absence and by 9 and 26% in the presence in media of HCO 3 − ion. It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive branch of the tricarboxylic acid cycle, is caused by activation of the glyoxylate shunt.
Heterogenous expression of poly-γ-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3 by N. Wang; G. Yang; C. Che; Y. Liu (pp. 381-385).
Bacillus licheniformis WBL-3, one of poly-γ-glutamic acid (γ-PGA) producers, depends on the existence of glutamate in the medium. In this paper, γ-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgs-BCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IFO3336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced γ-PGA extracellularly. The yield of γ-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar γ-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize γ-PGA.
Transformation of Δ4-3-ketosteroids by free and immobilized cells of Rhodococcus erythropolis actinobacterium by N. V. Carpova-Rodina; V. A. Andryushina; V. V. Yaderetz; A. V. Druzhinina; T. S. Stytsenko; B. L. Shaskol’skiy; V. I. Lozinsky; Luu Duc Huy; N. E. Voishvillo (pp. 386-392).
9α-Hydroxy derivatives were prepared from 11 steroids of androstane and pregnane series using Rhodococcus erythropolis VKPM Ac-1740 culture with 0.5–10 g/l substrate concentration in the reaction mixture. 9α-Monohydroxylation proceeded regardless of the substituent structure at C17. However, the structure of the steroid molecule influenced the time of complete conversion of the substrate and the yield of the transformation product. 9α-Hydroxy-androstenedione was obtained in 35 h in a yield of 85% when the maximum concentration of androstenedione (AD) was 10 g/l. 9α-Hydroxy-AD was also formed by the actinobacterium cells entrapped in poly(vinyl alcohol) cryogel beads. Nine successive transformation cycles were carried out using immobilized cells at 4.0 g/l concentration of AD in the medium. The yield of 9α-hydroxy-AD formed during six cycles (from two to eight with the duration of each cycle for 22–24 h) was 98%.
Scaling of the process of biosynthesis of surfactants by Rhodococcus erythropolis EK-1 on hexadecane by T. P. Pirog; S. V. Ignatenko (pp. 393-399).
Peculiarities of synthesis of surface-active substances (SAS) are studied at periodical cultivation of Rhodococcus erythropolis EK-1 in the AK-210 fermenter on medium containing n-hexadecane. Maximum indicators of SAS synthesis (concentration of extra cellular SAS is 7.2 g/l; factor of emulsification of the cultural liquid 50%; SAS yield from the substrate 50%) have been observed at 60–70% concentration of dissolved oxygen from the saturation level with aerial oxygen (pH 8.0) fractional supply of the substrate by portions each being 0.3–0.4% every 5–6 h to a final volume concentration of 2.4% and with the use of 10% inoculate grown until mid-exponential phase on the medium with 1.0 vol % of n-hexadecane. Implementation of the process of SAS biosynthesis with the fermentation equipment provided the possibility to increase almost two-fold the amount of the synthesized SAS and reduce 3.5-fold the time of cultivation of the producer strain compared with the growth in flasks at shake-flask propagator.
Chemiluminescence analysis of oil oxidizing bacteria Actinetobacter calcoaceticus extracts: Effects of the extracts on pSoxS-lux biosensor by I. S. Sazykin; V. N. Prokofiev; V. A. Chistyakov; M. A. Sazykina; V. V. Vnukov (pp. 400-404).
A comparative H2O2-luminol- and Fe(II)-induced chemiluminescence analysis of extracts of two strains of marine oil oxidizing bacteria Actinetobacter calcoaceticus cultivated either in the presence or absence of oil was carried out. Effects of these extracts on E. coli MG1655 biosensor (pSoxS-lux) were studed. Activation of H2O2-induced chemiluminescence in the presence of oil was observed. This suggests activation of free radical lipid peroxidation. Aqueous extracts of microorganisms cultivated in the presence of oil were shown to activate reactive oxygen species production (ROS) in Fe(II)-induced chemiluminescence reaction mixture. Acetone-ethanol extracts induced antioxidative systems of both strains. Chemiluminescence analysis in a biological system carried utilizing E. coli MG1655 (pSoxS-lux) revealed that aqueous extracts of the strains cultivated in the absence of oil contained potential antioxidants.
Filamentous fungi’s cell-wall extraction at different stages of ontogenesis and exploration of their carbohydrate composition by D. A. Andriyanova; Ya. E. Sergeeva; G. A. Kochkina; L. A. Galanina; A. I. Usov; E. P. Feofilova (pp. 405-411).
Methods of obtaining cell walls (CW) for specimens of mucoraceous molds and ascomycetic affined fungi are developed at the stage of mycelium and resting cells, or spores. CW purity was assessed by electron microscopy, specific staining methods, scourage control, presence of ribose and desoxyribose, and the comparison of chitin content in whole cells and CW of fungi (a new criteria). The authors discuss the significance of the proposed methods of obtaining pure fractions of CW and of the study of their carbohydrate content for the chemotaxonomy of filamentous fungi.
An extracellular glucoamylase produced by endophytic fungus EF6 by P. Tangngamsakul; A. Karnchanatat; P. Sihanonth; P. Sangvanich (pp. 412-418).
A strain of endophytic fungus EF6 isolated from Thai medicinal plants was found to produce higher levels of extracellular glucoamylase. This strain produced glucoamylase of culture filtrate when grown on 1% soluble starch. The enzyme was purified and characterized. Purification steps involved (NH4)2SO4 precipitation, anion exchange, and gel filtration chromatography. Final purification fold was 14.49 and the yield obtained was 9.15%. The enzyme is monomeric with a molecular mass of 62.2 kDa as estimated by SDS-PAGE, and with a molecular mass of 62.031 kDa estimated by MALDI-TOF spectrometry. The temperature for maximum activity was 60°C. After 30 min for incubation, glucoamylase was found to be stable lower than 50°C. The activity decrease rapidly when residual activity was retained about 45% at 55°C. The pH optimum of the enzyme activity was 6.0, and it was stable over a pH range of 4.0–7.0 at 50°C. The activity of glucoamylase was stimulated by Ca2+, Co2+, Mg2+, Mn2+, glycerol, DMSO, DTT and EDTA, and strongly inhibited by Hg2+. Various types of starch were test, soluble starch proved to be the best substrate for digestion process. The enzyme catalyzes the hydrolysis of soluble starch and maltose as the substrate, the enzyme had K m values of 2.63, and 1.88 mg/ml and V max, values of 1.25, and 2.54 U/min/mg protein, and V max/K m values of 0.48 and 1.35, respectively. The internal amino acid sequences of endophytic fungus EF6 glucoamylase; RALAN HKQVV DSFRS have similarity to the sequence of the glucoamylase purified form Thermomyces lanuginosus. From all results indicated that this enzyme is a glucoamylase (1,4-α-D-glucan glucanohydrolase).
Antioxidant components of Laetiporus sulphureus (Bull.: Fr.) Murr. fruit bodies by D. N. Olennikov; L. M. Tankhaeva; S. V. Agafonova (pp. 419-425).
Antioxidant activity of fruit bodies of Laetiporus sulphureus (Bull.: Fr.) Murr. (Polyporales) obtained by the natural plantation growing method in Pribaikal’e (Irkutsk region) has been studied. It was determined that the ethyl acetate fraction of L. sulphureus, which was chromatographically separated into seven compounds identified as quercetin, kaempferol, (+)-catechin, p-coumaric, gallic, caffeic, and chlorogenic acids was characterized with more expressed antioxidant activity. All compounds were extracted from this basidiomycete species for the first time. The quantitative amount of the substances isolated from L. sulphureus was determined by HPLC. It was found that antioxidant activity of preparations obtained from L. sulphureus is conditioned by phenolic compounds.
Physiological and biochemical characteristics of the genus Penicillium fungi as producers of ergot alkaloids and quinocitrinins by A. G. Kozlovsky; V. P. Zhelifonova; T. V. Antipova; N. F. Zelenkova (pp. 426-430).
Four cultures of fungi of the genus Penicillium belonging to Furcatum Pitt subgenus, such as P. citrinum Thom, 1910; P. corylophilum Dierckx, 1901; P. fellutanum Biourge, 1923; and P. waksmanii Zaleski, 1927, produced the ergot alkaloids, namely, agroclavine-I, and epoxyagroclavine-I; their N-N-dimers, such as dimer of epoxyagroclavine-I and the mixed dimer of epoxyagroclavine-I and agroclavine-I; and also quinoline metabolites, namely, quinocitrinin A and quinocitrinin B. Physiological and biochemical characteristics of the producers were studied. Optimal conditions for the biosynthesis of metabolome components were determined. Zinc additive to the medium stimulated the biosynthesis of the ergot alkaloids in all cases; quinocitrinines production was increased only in P. citrinum, and that was suppressed in P. corylophinum, P. fellutanum, and P. waksmanii. This testifies that genes of the biosynthesis pathways are located in the different clusters of the producers.
Influence of salicylic acid on biosynthesis of nucleic acids in Polyscias filicifolia cell culture under the action of unfavorable temperatures by N. V. Kirillova; Yu. V. Belykh; A. I. Spasenkov (pp. 431-434).
Amounts of DNA and RNA was increased (from 20 to 50%) in the presence of salicylic acid in cells of Polyscias filicifolia tissue culture grown in Murachige-Skoog modified medium. Treatment of the tissue culture with salicylic acid resulted in a significant increase of intracellular protein and decrease of proteolytic activity. In cells treated with salicylic acid, the amounts of DNA and RNA was higher in conditions of heat (3 h, 45°C) and cold (24 h, 7°C) stress in comparison with cells exposed to unfavorable temperatures without the initial treatment with salicylic acid.
Effect of salicylic acid on the proton translocation activity of plasmalemma of potato tuber cells by E. P. Ladyzhenskaya; N. P. Korablyova (pp. 435-439).
Action of salicylic acid (SA) on the activity of membrane bound H+-ATPase and passive proton permeability of plasmalemma membrane vesicles (PMV) from parenchyma cells of potato tubers was detected. A correlation between SA action on germination of tubers and activity of plasmalemma H+-ATPase was revealed: the application of growth-stimulating concentrations of SA (10−10–10−8 M) in the system in vitro resulted in activation of plasmalemma H+-ATPase, while the use of growth-inhibiting concentrations (10−4, 10−5 M) provoked inhibition of the enzyme activity. Addition of jasmonic acid (JA) to the incubation mix resulted in increase of SA effect on the accumulation of H+ in PMV.
Effect of melafen on mitochondrial apparatus of apical meristem in growth regulation in potato tubers by T. A. Platonova; A. S. Evsyunina; N. P. Korablyova (pp. 440-444).
Growth stimulation in potato Solanum tuberosum L. tubers by melafen preparation caused an increase in area of mitochondrial apparatus (increase in mitochondrial size) in apical meristem cells. Melafen stimulated mitochondrial differentiation (increase in number of condensed mitochondria enriched in cristas). Obtained data revealed an increase in activity of mitochondrial apparatus which is connected with an increase in energetic demands of cells in potato tuber apexes at melafen growth activation.
Oregano essential oil as an inhibitor of higher fatty acid oxidation by M. B. Terenina; T. A. Misharina; N. I. Krikunova; E. S. Alinkina; L. D. Fatkulina; A. K. Vorob’yova (pp. 445-449).
Inhibition of the oxidation of fatty acids methyl esters by oregano essential oil was studied using capillary gas chromatography. A mixture of fatty acids which contained saturated, mono-, di-, and polyunsaturated acids with 16–24 carbon atoms was extracted from mice brain. Changes in the composition of esters in hexane solutions both in the presence of oregano essential oil and without it were examined during their autooxidation in light for 1 year. It was found that the oxidation rate of unsaturated fatty acids increases with increasing degree of their unsaturation. Oregano essential oil inhibited the oxidation process. Antioxidant activity of the oil increased with increase of its concentration. It was shown that carvacrol and thymol are the main antioxidant components of oregano oil.